Molecular beacons were introduced in the mid-1990's as novel probes that can fluorescently detect in solution or in living cells nucleic acid synthesis, expression or trafficking. The basic structure of molecular beacons includes a stem-loop structure with a fluorophore and a quencher at the respective 5′ and 3′ ends of the molecule. Proximity of the fluorophore to the quencher results in fluorescence resonance energy transfer and quenching of the fluorescence. Upon hybridization of the loop region to a target DNA or RNA in solution or in living cells, the fluorophore and quencher become spatially separated resulting in emission of fluorescence. Molecular beacons have been widely used to detect either DNA or RNA in the real-time quantitative PCR methodologies. Microinjection of antisense oligonucleotide molecular beacons designed to detect the myb/vav protooncogene in human K562 leukemia cells provided evidence for DNA-RNA hybridization in living cells.
One of the potential targets for real-time in vivo imaging with molecular beacons is the p53 pathway which, since dysfunctional in cancer, portends a poor patient prognosis and poor response to chemo- and radiotherapy. Mutations in the p53 gene are common in human tumors and even when the gene is wild-type there are usually other defects resulting in p53 inactivity. Mechanisms of p53 inactivation include accelerated degradation due to MDM2 or human papillomavirus E6 protein, cytoplasmic sequestration due to Parc, inefficient stabilization due to ARF deletion or mutation, or dominant negative isoforms of p53 family members. While detection of p53 mutation in vivo may be potentially useful in prognostication, this has proven to be difficult for a variety of reasons.
p53 mutations occur at many different sites primarily within a large central DNA-binding domain, and while it is possible to detect such mutations with microarray technology this requires DNA isolation. Immunohistochemistry has been used to detect overexpressed p53 protein due to the fact that most mutants have a greatly increased half-life. However, increased expression of p53 occurs even when p53 is wild-type and so this has not been a uniformly reliable method. While there are some antibodies that can distinguish wild-type from some mutated epitopes of p53, these methods require at a minimum fixing the cells and therefore cannot be used in living tissue.
In the last decade, significant progress has been made in the identification and characterization of the events downstream of p53 activation. Wild-type p53 protein is stabilized in response to a variety of cellular stresses including exposure to DNA damaging chemo- or radio-therapy, hypoxia, or inappropriate proliferative signals such as oncogene activation. Transcriptional targets of p53 have been linked to its tumor suppressive activities. For example, the p53-dependent transcriptional activation of p21(WAF1/CIP1) in response to cellular stress results in cell cycle arrest through inhibition of cyclin-dependent kinase activity.5,6 Other p53 targets include death receptors such as Fas or KILLER/DR5, or other proapoptotic genes such as Puma, Noxa, Bax, Bak, Bid, or PIDD that either directly or indirectly impact on the mitochondria or caspase activation leading to cell death. The most consistently induced p53 target gene is the p21(WAF1/CIP1) gene, being induced during either cell cycle arrest or apoptosis or in all tissues or cell lines examined.
Therefore, there is a need to non-invasively imaging cellular and molecular events associated with effective chemotherapy and radiotherapy that use p21(WAF1/CIP1) gene.